Dynamics of proteins in Golgi membranes: comparisons between mammalian and plant cells highlighted by photobleaching techniques

In less than a decade the green fluorescent protein (GFP) has become one of the most popular tools for cell biologists for the study of dynamic processes in vivo. GFP has revolutionised the scientific approach for the study of vital organelles, such as the Golgi apparatus. As Golgi proteins can be tagged with GFP, in most cases without altering their targeting and function, it is a great substitute to conventional dyes used in the past to highlight this compartment. In this review, we cover the application of GFP and its spectral derivatives in the study of Golgi dynamics in mammalian and plant cells. In particular, we focus on the technique of selective photobleaching known as fluorescence recovery after photobleaching, which has successfully shed light on essential differences in the biology of the Golgi apparatus in mammalian and plant cells.     

Yeast Golgi apparatus – dynamics and sorting

The yeast Saccharomyces cerevisiae is one of the best-studied organisms to understand molecular mechanisms of membrane traffic, but as far as the organization of the Golgi apparatus is concerned, yeast is only just beginning to yield clues about how dynamic and flexible the organelle is.     

Membrane traffic in the secretory pathway

The steady-state localisation of membrane proteins in the endocytic system is the result of many sorting events that occur at various points throughout the endosomal pathway. A protein that has been endocytosed from the plasma membrane or sorted at the trans-Golgi network (TGN) and transported to an endosome will ultimately be delivered to one of three destinations: the plasma membrane, the TGN or the lysosome. Where a membrane protein is trafficked to depends on the interactions between sorting motifs present in the membrane protein and the machinery that can decode these motifs. Much of the protein machinery that recognises sorting motifs is conserved from yeast toman, and in this review I will discuss this machinery and the motifs that govern endosomal protein sorting. (Part of a Multi-author Review)     

Are Rab proteins the link between Golgi organization and membrane trafficking?

The fundamental separation of Golgi function between subcompartments termed cisternae is conserved across all eukaryotes. Likewise, Rab proteins, small GTPases of the Ras superfamily, are putative common coordinators of Golgi organization and protein transport. However, despite sequence conservation, e.g., Rab6 and Ypt6 are conserved proteins between humans and yeast, the fundamental organization of the organelle can vary profoundly. In the yeast Saccharomyces cerevisiae, the Golgi cisternae are physically separated from one another, while in mammalian cells, the cisternae are stacked one upon the other. Moreover, in mammalian cells, many Golgi stacks are typically linked together to generate a ribbon structure. Do evolutionarily conserved Rab proteins regulate secretory membrane trafficking and diverse Golgi organization in a common manner? In mammalian cells, some Golgi-associated Rab proteins function in coordination of protein transport and maintenance of Golgi organization. These include Rab6, Rab33B, Rab1, Rab2, Rab18, and Rab43. In yeast, these include Ypt1, Ypt32, and Ypt6. Here, based on evidence from both yeast and mammalian cells, we speculate on the essential role of Rab proteins in Golgi organization and protein transport.     

ER-to-Golgi transport and cytoskeletal interactions in animal cells

The endoplasmic reticulum (ER)-Golgi system has been studied using biochemical, genetic, electron and light microscopic techniques. We now understand many aspects of trafficking from the ER to the Golgi apparatus, including some of the signals and mechanisms for selective retention and retrieval of ER resident proteins and export of cargo proteins. Proteins that leave the ER emerge in export complexes or ER exit sites and accumulate in pleiomorphic transport carriers referred to sometimes as VTCs or intermediate compartments. These structures then transit from the ER to the Golgi apparatus along microtubules using the dynein/dynactin motor and fuse with the cis cisterna of the Golgi apparatus. Many proteins (including vSNAREs, ERGIC53/p58 and the KDEL receptor) must cycle back to the ER from pre-Golgi intermediates or the Golgi. We will discuss both the currently favored model that this cycling occurs via 50-nm COPI-coated vesicles and in vivo evidence that suggests retrograde trafficking may occur via tubular structures.     

Dissection of a novel molecular determinant mediating Golgi to trans-Golgi network transition

Two major functions of the Golgi apparatus (GA) are formation of complex glycans and sorting of proteins destined for various subcellular compartments or secretion. To fulfill these tasks proper localization of the accessory proteins within the different sub-compartments of the GA is crucial. Here we investigate structural determinants mediating transition of the two glycosyltransferases β-1,4- galactosyltransferase 1 (gal-T1) and the α-1,3-fucosyltransferase 6 (fuc-T6) from the trans-Golgi cisterna to the trans-Golgi network (TGN). Upon treatment with the ionophore monensin both glycosyltransferases are found in TGN-derived swollen vesicles, as determined by confocal fluorescence microscopy and density gradient fractionation. Both enzymes carry a signal consisting of the amino acids E₅P₆ in gal-T1 and D₂P₃ in fuc-T6 necessary for the transition of these glycosyltransferases from the trans-Golgi cisterna to the TGN, but not for their steady state localization in the trans-Golgi cisterna. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 30 July 2008; received after revision 17 September 2008; accepted 29 September 2008     

Membrane traffic in the secretory pathway

It is generally thought that microtubule-associated motors insure long-range movements of the secretory vesicles from the center of the cell to its periphery, while myosins insure short-range movements at the cell periphery. However, several of the myosins that have been reported during the last decade to be involved in the exocytic pathway are not processive, meaning that they do not have the ability to move cargos along actin polymers. We will review here the possible mechanisms by which these myosins could contribute to the traffic of secretory proteins from the Golgi complex to the plasma membrane.     

Trans-Golgi network sorting

The trans-Golgi network (TGN) is a major secretory pathway sorting station that directs newly synthesized proteins to different subcellular destinations. The TGN also receives extracellular materials and recycled molecules from endocytic compartments. In this review, we summarize recent progress on understanding TGN structure and the dynamics of trafficking to and from this compartment. Protein sorting into different transport vesicles requires specific interactions between sorting motifs on the cargo molecules and vesicle coat components that recognize these motifs. Current understanding of the various targeting signals and vesicle coat components that are involved in TGN sorting are discussed, as well as the molecules that participate in retrieval to this compartment in both yeast and mammalian cells. Besides proteins, lipids and lipid-modifying enzymes also participate actively in the formation of secretory vesicles. The possible mechanisms of action of these lipid hydrolases and lipid kinases are discussed. Finally, we summarize the fundamentally different apical and basolateral cell surface delivery mechanisms and the current facts and hypotheses on protein sorting from the TGN into the regulated secretory pathway in neuroendocrine cells. Received 2 November 2000; received after revision 19 February 2001; accepted 19 February 2001     

Membrane functional organisation and dynamic of μ-opioid receptors

The activation and signalling activity of the membrane μ-opioid receptor (MOP-R) involve interactions among the receptor, G-proteins, effectors and many other membrane or cytosolic proteins. Decades of investigation have led to identification of the main biochemical processes, but the mechanisms governing the successive protein–protein interactions have yet to be established. We will need to unravel the dynamic membrane organisation of this complex and multifaceted molecular machinery if we are to understand these mechanisms. Here, we review and discuss advances in our understanding of the signalling mechanism of MOP-R resulting from biochemical or biophysical studies of the organisation of this receptor in the plasma membrane.     

Scaffolds,levers, rods and springs: diverse cellular functions of long coiled-coil proteins

Long alpha-helical coiled-coil proteins are involved in a variety of organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems, motors, levers, rotating arms and possibly springs. A growing number of human diseases are found to be caused by mutations in long coiled-coil proteins. This review summarizes our current understanding of the multifaceted group of long coiled-coil proteins in the cytoskeleton, nucleus, Golgi and cell division apparatus. The biophysical features of coiled-coil domains provide first clues toward their contribution to the diverse protein functions and promise potential future applications in the area of nanotechnology. Combining the power of fully sequenced genomes and structure prediction algorithms, it is now possible to comprehensively summarize and compare the complete inventory of coiled-coil proteins of different organisms.Received 27 January 2004; received after revision 23 February 2004; accepted 10 March 2004     

The role of the <Emphasis Type="Italic">trans</Emphasis>-Golgi network in varicella zoster virus biology

The task of assembling nascent virions presents a formidable challenge to large, enveloped DNA viruses such as varicella zoster virus (VZV). After parasitising the host cells compartmentalised biosynthetic machinery, viral constituents must be brought together in appropriate proportions for packaging and export. Recent evidence places the trans-Golgi network (TGN) in an orchestrating role with respect to the assembly, envelopment and egress of herpesviruses. This role accords with known functions of the TGN in the uninfected cell. The targeting of viral glycoproteins to the TGN appears to provide a crucial platform for viral assembly. Tegument proteins, interacting with the cytoplasmic domains of glycoproteins, in turn recruit nucleocapsids to the developing supramolecular array. Molecular studies are continually refining understanding of these processes, building upon elegant electron microscopic data. Knowledge of VZVs use of endogenous trafficking pathways from the TGN sheds light on important aspects of viral behaviour in vitro and in vivo.Received 22 June 2004; received after revision 22 August 2004; accepted 25 August 2004     

Triple arginines in the cytoplasmic tail of endomannosidase are not essential for type II membrane topology and Golgi localization

Endomannosidase is a Golgi-localized endoglycosidase, which provides an alternate glucosidase-independent pathway of glucose trimming. Using a protease protection assay we demonstrated that Golgi-endomannosidase is a type II membrane protein. The first 25 amino acids of this protein, containing the cytoplasmic tail and the transmembrane domain, were sufficient for Golgi retention of fused reporter proteins alpha1-antitrypsin or green fluorescent protein. However, shortening or deletion of the transmembrane domain prevented Golgi localization, while lengthening it partially reduced Golgi retention of the enzyme. Substitution of the highly conserved positively charged amino acids within the cytoplasmic tail had neither an effect on type II topology nor on the inherent Golgi localization of the enzyme. In contrast, cytoplasmic tail-deleted rat endomannosidase possessed an inverted topology resulting in endoplasmic reticulum mislocalization. Thus, proper topology rather than the presence of positively charged amino acids in the cytoplasmic tail is critical for Golgi localization of rat endomannosidase.     

Localization of Golgi-resident glycosyltransferases

For many glycosyltransferases, the information that instructs Golgi localization is located within a relatively short sequence of amino acids in the N-termini of these proteins comprising: the cytoplasmic tail, the transmembrane spanning region, and the stem region (CTS). Also, one enzyme may be more reliant on a particular region in the CTS for its localization than another. The predominance of these integral membrane proteins in the Golgi has seen these enzymes become central players in the development of membrane trafficking models of transport within this organelle. It is now understood that the means by which the characteristic distributions of glycosyltransferases arise within the subcompartments of the Golgi is inextricably linked to the mechanisms that cells employ to direct the flow of proteins and lipids within this organelle.     

Cytoskeleton-mediated templating of complex cellulose-scaffolded extracellular structure and its association with oikosins in the urochordate <Emphasis Type="Italic">Oikopleura</Emphasis>

Oriented cellulose deposition is critical to plant patterning and models suggest microtubules constrain cellulose synthase movements through the plasma membrane. Though widespread in plants, urochordates are the only animals that synthesize cellulose. We characterized the distinctive cellulose microfibril scaffold of the larvacean house and its interaction with house structural proteins (oikosins). Targeted disruption of cytoskeletal elements, secretory pathways, and plasma membrane organization, suggested a working model for templating extracellular cellulose microfibrils from animal cells that shows both convergence and differences to plant models. Specialized cortical F-actin arrays template microfibril orientation and glycosylphosphatidylinositol-anchored proteins in lipid rafts may act as scaffolding proteins in microfibril elongation. Microtubules deliver and maintain cellulose synthase complexes to specific cell membrane sites rather than orienting their movement through the membrane. Oikosins are incorporated into house compartments directly above their corresponding cellular field of expression and interact with the cellulose scaffold to a variable extent.     

High affinity recognition of a <Emphasis Type="Italic">Phytophthora</Emphasis> protein by <Emphasis Type="Italic">Arabidopsis</Emphasis> via an RGD motif

The RGD tripeptide sequence, a cell adhesion motif present in several extracellular matrix proteins of mammalians, is involved in numerous plant processes. In plant-pathogen interactions, the RGD motif is believed to reduce plant defence responses by disrupting adhesions between the cell wall and plasma membrane. Photoaffinity cross-linking of [¹²⁵I]-azido-RGD heptapeptide in the presence of purified plasma membrane vesicles of Arabidopsis thaliana led to label incorporation into a single protein with an apparent molecular mass of 80 kDa. Incorporation could be prevented by excess RGD peptides, but also by the IPI-O protein, an RGD-containing protein secreted by the oomycete plant pathogen Phytophthora infestans. Hydrophobic cluster analysis revealed that the RGD motif of IPI-O (positions 53–56) is readily accessible for interactions. Single amino acid mutations in the RGD motif in IPI-O (of Asp⁵⁶ into Glu or Ala) resulted in the loss of protection of the 80-kDa protein from labelling. Thus, the interaction between the two proteins is mediated through RGD recognition and the 80-kDa RGD-binding protein has the characteristics of a receptor for IPI-O. The IPI-O protein also disrupted cell wall-plasma membrane adhesions in plasmolysed A. thaliana cells, whereas IPI-O proteins mutated in the RGD motif (D56A and D56E) did not.Received 23 October 2003; received after revision 5 December 2003; accepted 12 December 2003     

Cellular microbiology of intracellular <Emphasis Type="Italic">Salmonella enterica</Emphasis>: functions of the type III secretion system encoded by <Emphasis Type="Italic">Salmonella</Emphasis> pathogenicity island 2

The facultative intracellular pathogen Salmonella enterica resides in a special membrane compartment of the host cell and modifies its host to achieve intracellular survival and proliferation. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has a central role in the interference of intracellular Salmonella with host cell functions. SPI2 function affects antimicrobial defense mechanisms of the host, intracellular transport processes, integrity and function of the cytoskeleton and host cell death. These modifications are mediated by translocation of a large number of effector proteins by the SPI2 system. In this review, we summarize recent work on the cellular phenotypes related to SPI2 function and contribution of SPI2 effector proteins to these manipulations. These studies reveal a complex set of pathogenic interferences between intracellular Salmonella and its host cells.Received 11 June 2004; received after revision 8 July 2004; accepted 12 July 2004     

The FN3 and BRCT motifs in the exomer component Chs5p define a conserved module that is necessary and sufficient for its function

Chs5p is a component of the exomer, a coat complex required to transport the chitin synthase Chs3p from the trans-Golgi network to the plasma membrane. The Chs5p N-terminal region exhibits fibronectin type III (FN3) and BRCT domains. FN3 domains are present in proteins that mediate adhesion processes, whereas BRCT domains are involved in DNA repair. Several fungi—including Schizosaccharomyces pombe, which has no detectable amounts of chitin—have proteins similar to Chs5p. Here we show that the FN3 and BRCT motifs in Chs5p behave as a module that is necessary and sufficient for Chs5p localization and for cargo delivery. The N-terminal regions of S. cerevisiae Chs5p and S. pombe Cfr1p are interchangeable in terms of Golgi localization, but not in terms of exomer assembly, showing that the conserved function of this module is protein retention in this organelle and that the interaction between the exomer components is organism-specific.     

The presynaptic cytomatrix of brain synapses

Synapses are principal sites for communication between neurons via chemical messengers called neurotransmitters. Neurotransmitters are released from presynaptic nerve terminals at the active zone, a restricted area of the cell membrane situated exactly opposite to the postsynaptic neurotransmitter reception apparatus. At the active zone neurotransmitter-containing synaptic vesicles (SVs) dock, fuse, release their content and are recycled in a strictly regulated manner. The cytoskeletal matrix at the active zone (CAZ) is thought to play an essential role in the organization of this SV cycle. Several multi-domain cytoskeleton-associated proteins, including RIM, Bassoon, Piccolo/Aczonin and Munc-13, have been identified, which are specifically localized at the active zone and thus are putative molecular components of the CAZ. This review will summarize our present knowledge about the structure and function of these CAZ-specific proteins. Moreover, we will review our present view of how the exocytotic and endocytic machineries at the site of neurotransmitter release are linked to and organized by the presynaptic cytoskeleton. Finally, we will summarize recent progress that has been made in understanding how active zones are assembled during nervous system development.     

Membrane shaping by the Bin/amphiphysin/Rvs (BAR) domain protein superfamily

BAR domain superfamily proteins have emerged as central regulators of dynamic membrane remodeling, thereby playing important roles in a wide variety of cellular processes, such as organelle biogenesis, cell division, cell migration, secretion, and endocytosis. Here, we review the mechanistic and structural basis for the membrane curvature-sensing and deforming properties of BAR domain superfamily proteins. Moreover, we summarize the present state of knowledge with respect to their regulation by autoinhibitory mechanisms or posttranslational modifications, and their interactions with other proteins, in particular with GTPases, and with membrane lipids. We postulate that BAR superfamily proteins act as membrane-deforming scaffolds that spatiotemporally orchestrate membrane remodeling.     

Molecular mechanisms of centrosome and cytoskeleton anchorage at the nuclear envelope

Cell polarization is a fundamental process underpinning organismal development, and tissue homeostasis, which requires an orchestrated interplay of nuclear, cytoskeletal, and centrosomal structures. The underlying molecular mechanisms, however, still remain elusive. Here we report that kinesin-1/nesprin-2/SUN-domain macromolecular assemblies, spanning the entire nuclear envelope (NE), function in cell polarization by anchoring cytoskeletal structures to the nuclear lamina. Nesprin-2 forms complexes with the kinesin-1 motor protein apparatus by associating with and recruiting kinesin light chain1 (KLC1) to the outer nuclear membrane. Similar to nesprin-2, KLC1 requires lamin A/C for proper NE localization. The depletion of nesprin-2 or KLC1, or the uncoupling of nesprin-2/SUN-domain protein associations impairs cell polarization during wounding and dislodges the centrosome from the NE. In addition nesprin-2 loss has profound effects on KLC1 levels, the cytoskeleton, and Golgi apparatus organization. Collectively these data show that NE-associated proteins are pivotal determinants of cell architecture and polarization.